Diazepam metabolism by human liver microsomes is mediated by both S-mephenytoin hydroxylase and CYP3A isoforms.

1. The primary metabolism of diazepam was studied in human liver microsomes in order to investigate the kinetics and to identify the cytochrome P450 (CYP) isoforms responsible for the formation of the main diazepam metabolites, temazepam and N-desmethyldiazepam. 2. The formation kinetics of both metabolites were atypical and consistent with the occurrence of substrate activation. A sigmoid Vmax model equivalent to the Hill equation was used to fit the data. The degree of sigmoidicity was greater for temazepam formation than for N-desmethyldiazepam formation, so that the ratio of desmethyldiazepam:temazepam formation increased as the substrate (diazepam) concentration decreased. 3. alpha-Naphthoflavone activated both reactions but with a greater effect on temazepam formation than on N-desmethyldiazepam formation. In the presence of 25 microM alpha-naphthoflavone the kinetics for both pathways were approximated by Michaelis-Menten kinetics. 4. Studies with a series of CYP isoform selective inhibitors and with an inhibitory anti-CYP2C antibody indicated that temazepam formation was carried out mainly by CYP3A isoforms, whereas the formation of N-desmethyldiazepam was mediated by both CYP3A isoforms and S-mephenytoin hydroxylase.